(1) Field of the Invention
New tylosin derivatives having at least one acyl group at the 3- and 4"-positions of tylosin, and the acid addition salts thereof, which inhibit the growth of various microorganisms including drug-resistant bacterial isolants and which produce high blood levels through oral administration are produced by a biochemical reaction using the microorganisms of the genus Streptomyces which are selected for their newly-found ability to acylate the 3- and 4"-positions of 16-membered macrolide antibiotics; they are recovered from the reacted mixture by conventional methods for recovering macrolide antibiotics. The 3- and 4"-positions of 16-membered macrolide antibiotics in this specification mean the 3-position of 16-membered ring and the 4"-position of mycarose of the macrolide antibiotic, respectively.
(2) Characteristics of the Invention
Acylation of antibiotics is one of the practical methods for the production of new antibiotic species and their derivatives. Chemical synthesis is commonly employed for such acylation, but the acylation is liable to occur in a uniform fashion in many hydroxyl groups, whereby further reaction steps are required to obtain the desired product which is to be acylated at particular positions. Biochemical processes, on the other hand, facilitate selective acylation only at target positions due to the specificity of enzyme reactions and the yield is usually high. The present inventors have made a thorough investigation of one such biochemical conversion of macrolide antibiotics, especially of the acylation of macrolide antibiotics, and have found a number of microorganisms which are capable of specifically acylating the 3- and 4"-positions of 16-membered macrolide antibiotics. This is the first and novel finding of an enzyme reaction of the aforementioned type originating from microorganisms, and it is particularly applied to the production of new tylosin derivatives from tylosin.
(3) Description of the Prior Art
There has been no prior description regarding the tylosin compounds of the present invention, which are produced by the biochemical acylation of tylosin.
The chemical synthesis of tylosin acetyl ester and of acetylated tylosin is described in Japanese Patent (Kokoku) Showa No. 36-22649 entitled "Method for Producing Tylosin" as described in Examples 4 and 5. However, the products are obviously different from the tylosin derivatives of the present invention, for the reason presented in the Detailed Description hereinbelow.
(4) Characteristics of the Process of the Invention
The acylation of antibiotics is one of the practical methods for the production of new antibiotic species and derivatives. Chemical synthesis is commonly employed for such acylation, but the acylation is liable to occur in a uniform fashion throughout the hydroxyl groups of the compound, whereby extra measures and steps are required, e.g., the proper protection of some hydroxyl groups and of other functional residues, to obtain the desired product which is acylated at one particular position. A biochemical process, on the other hand, facilitates selective acylation due to the specificity of enzyme reaction and the yield is usually high. The present inventors have made a thorough investigation of such biochemical transformation of macrolide antibiotics, and have found that a number of microorganisms are capable of specifically and simultaneously acylating the 3- and 4"-positions of 16-membered macrolide antibiotics. A particular feature of this process is that a variety of acylated derivatives can be produced by the proper combination of the two components of the acylation systems produced by one organism, namely, the substrate antibiotics and the acyl donors, which is of substantial industrial merit.
There are two prior examples reported in connection with processes for the microbial acylation of 16-membered macrolide antibiotics, i.e., of spiramycin and YL-704. The process using spiramycin is presented in U.S. Pat. No. 2,943,024, entitled "Preparation of Spiramycin III", U.S. Pat. No. 2,943,025, entitled "Preparation of Spiramycin II", French Patent 1,262,571, entitled "Transformation Biochemique de la Spiramycin I en Spiramycin II et III" and Japanese Patent (Kokoku) Showa No. 36-349, entitled "Process for Producing Spiramycin II, Spiramycin III and the Mixture Thereof." These patents practically deal with the same invention translated into different languages, describing the processes which may be numerized as follows;
1. A spiramycin-producing organism, of Streptomyces ambofaciens NRRL 2420 was cultivated in a culture medium to which spiramycin I, having a 3-hydroxyl group, was added and acylating agents, spiramycin II having a 3-acetyl group, and spiramycin III, having a 3-proionyl group, were produced.
2. The cells cultivated free of spiramycin and the acylating agents were suspended in a reaction medium, to which was added spiramycin I and the acylating agents. After incubation, spiramycin II and III were produced.
3. In the fermentational production of spiramycins with the aforementioned organism, the addition of the acylating agents enhanced the production ratio of spiramycin II or III.
The process according to the present invention is clearly distinguishable in principle from the prior art process for microbially acylating spiramycin for the following reasons.
1. The prior art process uses an organism which is a direct producer of spiramycin, and the process is closely linked to antibiotic fermentation. The four favorable strains used in the present invention are non-producers of the desired antibiotic species, and this indicates that the process of the present invention is an enzymatic process in principle.
2. The substrate antibiotic used in the prior art process is limited to spiramycin I, which is a direct product of the organism, of the prior art process, while the process of the present invention can employ most of the 16-membered macrolide antibiotics, indicating the non-specific nature of the reaction in terms of substrate specificity.
3. The prior art process can effect the conversion of only the 3-position of spiramycin I, while the process of the present invention can, if intended, carry out the simultaneous conversion of the 3- and 4"-positions with one organism. A much wider variety of products can be obtained with the proper combination of the substrate, the acyl donor and the reaction conditions. Also of significance is the simplicity of the process in converting the two functional positions simultaneously with one cell system.
With regard to YL-704, which is a family of leucomycins, Japanese Patent (Kokoku) Showa No. 49-13992 "Process for Producing an Antibiotic YL-704A.sub.1 " is presented. This patent indicates that an organism selected from the group consisting of Streptomyces eurocidicus NIHJ-267, Streptomyces albireticuli IFO-12737, Streptomyces kitasatoensis NRRL-2486 and Streptomyces sp. MCRL-0737 is cultivated in a medium containing "DHP compound", 4"-deacyl YL-704A.sub.1, or the DHP compound and L-leucine and after its cultivation, YL-704A.sub.1, which has an isovaleryl group at 4"-position of the DHP compound, is separated.
The processes of the present invention are also obviously different from that of the last mentioned patent, relating to YL-704A.sub.1 production, in respects like those described in the prior art case of the spiramycin process, namely with respect to the organisms employed, the enzymatic nature of the process of the present invention using enzyme system having no strict substrate specificity processes of the present invention, the variety of products, to be produced by the processes of the present invention and the fact that acylation at the 3- and 4"-positions can be effected simultaneously with one type of strain alloy which are of substantial industrial utility.